Basic DNA Testing Process and its Importance
Author: Cornelius Onye Nichodemus
DNA means Deoxyribonucleic acid is the genetic material of an organism. This plays a significant role in the genetic makeup of an individual thereby distinguishing one person from another. This unique difference that exists between individuals has resulted in DNA testing of individuals in order to determine the composition of the DNA which differs among humans. DNA testing involves a set of laboratory techniques required to isolate and study a given DNA sample. Basically, there are four(4) main stages involved in DNA testing, they are; Extraction, Quantitation, Amplification and Electrophoresis.
These involves isolating the DNA from nucleus of cells. DNA is located within the nucleus of cells throughout the body of an organism and the extraction step is responsible for the breaking open the nucleus and releasing the DNA molecules into solution. During this step, it is also possible to separate DNA molecules from all other cellular material and any other debris that may be present in a particular biological sample. Some of these materials can be potential inhibitors to steps later on in the DNA testing process. So it is important to try and isolate only the DNA molecules.
The extraction of DNA involves the following steps;
Step 1. Breaking cells open to release the DNA: the cells in the sample are separated from each other often by physical means such as grinding or vortexing and put into a solution containing salt. The positively charged Sodium ion in the salt help protect the negatively charged phosphate group that run along the backbone of the DNA. A detergent is added which breaks down the lipids in the cell membrane and nuclei. DNA is released as these membranes are disrupted.
Step 2. Separating DNA from proteins and other cellular debris: to get a clean sample of DNA, it's necessary to remove as much of the cellular debris as possible. This can be done by a variety of methods. Often a protease (protein enzyme) is added to degrade DNA associated proteins and other cellular proteins. Alternatively, some of the cellular debris can be removed by filtering the sample.
Step 3. Precipitating the DNA with an alcohol: finally, ice-cold alcohol (either ethanol or isopropanol) is carefully added to the DNA sample. DNA is soluble in water but insoluble in the presence of salt and alcohol. By gently stirring the alcohol layer with a sterile pipette, the precipitate becomes visible and can be spooled out. If there is lots of DNA, you can see a stingy white precipitate.
Step 4. Cleaning the DNA: the DNA sample can now be further purified (cleaned). It is then resuspended in a slightly alkaline buffer and ready to use.
Step 5. Confirming the presence and quality of the DNA: for further lab work, it is important to know the concentration and quality of the DNA.
Optical density readings taken by a spectrophotometer can be used to determine the concentration and purity of the DNA in a sample. Alternatively, gel electrophoresis can be used to show the presence of DNA in a sample give an indication of its quality.
More importantly, once the DNA is extracted, it can be used for molecular analysis including PCR, electrophoresis, sequencing, fingerprinting and cloning.
Is the process where the quality and quantity of DNA present in a sample is measured and assessed. Determining the amount of DNA in a sample is essential for success in the next step since most amplification systems require a small range of DNA input. Quantitation is one of the standards of all DNA testing lab required to ensure that the DNA recovered from an extraction is human rather than from other sources such as bacteria. Quantitation stage also involves digestion of the DNA with restriction enzymes that is cutting the DNA into specific pieces for the purpose of the study.
DNA amplification is achieved through the use of a technique called Polymerase Chain Reaction (PCR). PCR is a process in which millions of copies of a specific sequence of DNA can be made in a matter of only few hours for analysis. This is important for forensic DNA samples since the DNA often found at crime scenes is limited in both quality and quantity. In using a PCR, it is ideal to use a few DNA samples and set up the PCR machine before running the whole reactions.
4. CAPILLARY ELECTROPHORESIS (CE)
After the PCR reaction is completed it results in a large mixture of amplified DNA molecules which needs to be separated in order to distinguish the various molecules from one another. This is best done through a process known as electrophoresis. DNA molecules carry negative charge and once an electric current is applied to the sample, the molecules enter a very thin capillary filled with a gel-like polymer and migrate towards the positive anode at the other end of the capillary. The PCR products are then separated by size because the smaller DNA molecules will migrate easily through the polymer the larger DNA molecules. The presence of buffer solution in the electrophoresis makes the migration to the positive anode more easier. The data from this process is collected by placing the gel electrophoresis on a UV transilluminator connected to a computer. Through this, the image (DNA) is captured and further studies are carried out thereafter.
IMPORTANCE OF DNA TESTING
DNA testing plays an important role in research. It forms a basis for other fields such as medicine, agriculture, forensic science etc.
Forensic science: this is the DNA testing process to identify the individual involved in the crime scene. Investigators collect samples of blood, hair etc examine in the lab and identify the DNA of the person who committed the crime. This is used by the Bureau of Criminal Apprehension.
Agriculture: DNA testing has led to the genetic modification of important varieties of crops to make them more resistant to diseases and insects and also increase the crop yield. DNA testing and research is also used to improve the animal breeds. This genetic technique has led to more production of plants varieties.
Medicine: DNA testing is used in the following ways;
- Genetic research to trace the ancestorial origin of an individual
- Test to check or screen potential of a newborn baby
- DNA testing is used to know if an individual has a cancer or virus in his/her body
- To determine the biological father/mother of a child and it helps to solve legal cases.
About Author / Additional Info:
I am a First Class graduate of Plant Science and Biotechnology from University of port Harcourt.